The present invention relates to a device for selective exposure of a biological sample, preferably biological cell material or tissue samples, to sound waves, comprising a receptacle for the sample in which the biological sample is found in a suspended form and an electroacoustic transducer device which generates the sound waves and is disposed outside the receptacle for the sample in such a manner that sound-wave coupling occurs through the wall of the receptacle of the sample into the sample.
A multiplicity of applications of sound waves in the form of ultrasonic power for influencing the properties of a material or the structure of a material are known. For example, ultrasonic lithotriptors use ultrasound to crush kidney stones and gallstones. Furthermore, ultrasound is utilized for cleaning wounds and for phacoemulsification. In non-medical fields, ultrasound is employed, for example, for cleaning, boring, milling and welding.
Exposure of biological samples to ultrasound can trigger certain biological effects, for instance transportation of cells into cell reactors without impairing cell vitality, indeed destroying the cells, as is the case with the aforementioned lithotriptors. Due to the disintegrating effect on biological structures, ultrasonic emulisificators attained considerable significance in breaking down samples.
Such ultrasonic emulisifactors, so-called sonotrodes often have rapid transformers disposed on electroacoustic transducers such as for example Langevin""s or composite oscillators. The electroacoustic transducers frequently are provided with piezoceramic as the active material and oscillate with an amplitude of, for example, 0.5 xcexcm. With the aid of transformers, the initially very small amplitude is multiplied, for example, to over 500 xcexcm.
These ultrasonic emulsificators have proven themselves in practice and are often used. The effect of sound transmitted from the sonotrode to the biological sample depends on the sound pressure amplitude, which may vary strongly spatially due to the interference of sound waves reflected at the receptacle of the sample. As the temperature and location of the tip of the sonotrode in the sample strongly influence interferences and the sample usually warms up during treatment, it is difficult to dose ultrasound precisely for selective excitation of biological effects.
Furthermore, there is the danger of cross contamination from dipping the sonotrode in the sample if the sonotrode is not cleaned with the necessary care when changing samples.
The traditional design of the device does not prevent the foam or aerosol formation, which reduces the reproducibility of sound-wave exposure.
As, for electroacoustic reasons, sonotrodes cannot be designed as small as possible, there are limits to treating miniaturized samples such as are, e.g., used in breaking down samples for genetic analyses.
DE 32 09 841 C2 describes an arrangement for embedding at least one tissue sample in paraffin. The device provides a receptacle into which various coupling liquids can be introduced into which a certain sample of tissue to be treated is placed. To accelerate the willingness of the sample tissue to react with the respective liquids inside the receptacle, the device provides on the bottom of the receptacle an ultrasound generator 61, which couples ultrasonic waves with a frequency of 35 KHz into the interior of the work receptacle. However, with the selected frequencies, ultrasonic energy cannot be concentrated on very small sample volumes. Moreover, the device known from the art provides for disposing the ultrasound generating unit both inside and outside the sample receptacle. Therefore, it can be assumed that, in this case, there is no cross contamination problem.
The desire for concentrated introduction of ultrasonic waves in, for example, biological material is borne by the attempt to generate the desired process respectively reactions inside the cell material. A typical example of such a process is breaking down cells in order to examine the interior of the cells in subsequent detection processes.
As mentioned in the preceding, for breaking down the cell, ultrasonic waves which generate shear forces due to the biological cells in a suspension are employed, among other things, thereby breaking open the cell walls. In order to improve the effect of breaking open the cell, measures are undertaken to enhance the shear forces inside the suspension. For example, additional macroscopic particles, preferably in the form of small glass beads, are introduced into the suspension in order to enhance the action of the shear forces on the individual cells responsible for breaking them up.
In addition to the desire to enhance the shear forces acting on the cells, there is the need to miniaturize such types of devices, which seems to be in contradiction with the desire to enhance the shear forces.
The object of the present invention is to provide a device for exposing biological samples, preferably biological cell material, to sound waves, permitting precise intensity dosage of sound waves for selective excitation of biological effects. Furthermore, measures should be undertaken to increase the energy input in relation to the limited sample volume. In addition to the aforementioned requirements, it should be possible to miniaturize the size of the device in order to be able to conduct a large number of sample tests.
This object is solved with a device having the features of claim 1. Advantageous embodiments are described in the subclaims.
The invented device for selective exposure of a biological sample, preferably biological cell material, to sound waves, having a sample receptacle, in which the sample is in suspended form, and an electroacoustic transducer device, which is disposed outside the receptacle of the sample and which generates sound waves in such a manner that coupling the sound waves into the sample occurs through the wall of the sample receptacle, is further improved in such a manner that the electroacoustic transducer device generates sound waves having a frequency of at least 100 kHz, preferably of 500 kHz to 5 MHz, and that means for focusing the sound waves are provided which concentrate the sound waves approximately to 50 xcexcl on a sample volume inside the sample receptacle.
In the invented ultrasonic device, the electroacoustic transducer device interacts with the receptacle of the sample in such a manner that the ultrasonic coupling into the sampleoccurs through the wall of the receptacle of the sample and the electroacoustic transducer device is disposed outside the receptacle of the sample. As the sound, unlike in conventional ultrasonic emulsificators, is not radiated from the inside to the outside but from the outside to the inside, sound intensity distribution is limited to the sample so that in conjunction with a high mean frequency, lying in ranges of more than 100 kHz, the influence of the interference of the sound waves reflected at the wall of the receptacle for the sample can be controlled. This measure permits exact dosage of sonic intensity and precise excitation of certain biological effects in the sample volume down to 50 xcexcl and below. Typical sample volumes in which selective biological effects can be generated are approximately 100 xcexcl.
In an advantageous embodiment, sound coupling occurs through the floor of the sample receptacle in such a manner that the major part of the sound waves enters focused from below upward through the sample and is reflected at the top interface with reduced reflection at the wall of the sample receptacle. This is a very effective arrangement for controlling disturbing interferences.
For optimization of the intensity coupling, preferably a plane-parallel acoustic xcex/4 wave transformer is provided in the wall of the receptacle of the sample.
By inserting an acoustic lens in the wall of the receptacle of the sample, sound intensity distribution can be concentrated on a prescribed area of the volume in the sample receptacle.
The acoustic lens is preferably designed as a spherical acoustic lens and is insertable in the bottom of the sample receptacle or forms it completely.
Preferably sound-wave coupling occurs in the sample receptacle by means of structure-borne sound by providing a coupling element containing a soft polymer element or a liquid between the electroacoustic transducer device and the receptacle of the sample and being in immediate contact with the two or by providing a liquid coupling.
The invented selection of the used ultrasonic frequencies of at least 100 kHz, preferably 500 kHz to 5 MHz, permits focusing such high-frequency sound waves on the smallest areas, with their spatial sound intensity distribution being precisely influenceable. Thus, with such high-frequency sound waves, the area of maximum sound pressure can be focused on the center of the sample volume, thereby obviating spraying on the surface (aerosol formation) and therefore any cross contamination of adjacent samples.
Moreover, acoustic coupling at the wall of the receptacle of the sample by means of structure-borne sound is essentially free of losses so that small transducer devices suffice to generate sound. As they are disposed outside the sample receptacle, they can also be of very simple design in order to be able to provide the sound frequency required for the ultrasonic treatment of the biological samples. The invented ultransonic device can, therefore, be miniaturized and be employed with micro-titer plate systems.
Sound-wave coupling in the sample through the wall of the receptacle of the sample is not subject to changing parameters, which makes them exactly reproducible. Exact reproducibility of sound-wave coupling is a prerequisite for exact dosage and automated sample processing, in which the receptacle of the sample is automatically replaced respectively in which the biological sample in a sample receptacle is automatically exchanged.